Cell surface layer-binding protein and utilization thereof

ABSTRACT

A plasmid is constructed so as to express a fused protein of a sugar chain-binding protein domain with a desired protein. Then this plasmid is transferred into cells and thus the protein is expressed in the cell surface layer. This method is particularly adequate in case of expressing a protein having an activity in the C-terminal portion in cell surface layer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application of PCT/JP02/02801,entitled “Cell surface layer-binding protein and utilization thereof,”filed on Mar. 22, 2002, which claims the priority of Japanese PatentApplication No. 2001-12 1233, entitled “Cell surface layer-bindingprotein and utilization thereof,” filed on Apr. 19, 2001. The entirecontents and disclosure of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a cell surface layer-binding proteinhaving a sugar chain-binding protein domain, in which an additionalprotein is bound at least to the N-terminus or the C-terminus of thesugar chain-binding protein domain.

BACKGROUND ART

Cell surface layer-localized proteins are proteins that are present andimmobilized in a cell surface layer. An example thereof is α- ora-agglutinin, which is a flocculation protein of yeast. Such proteinsare similar to secretory proteins in that they have secretion signalsequences, but are different from secretory proteins in that they aretransported while being immobilized in a cell membrane via a GPI anchor.In general, cell surface layer-localized proteins have a GPI anchoringdomain on the C-terminal portion. Cell surface layer-localized proteinsare immobilized on a cell membrane in the following manner: when theprotein to be localized on the cell surface layer is passing across acell membrane, a part (i.e., a GPI anchor attachment recognition signalsequence) of the domain of the protein is selectively cleaved, a newlyprojected C-terminal portion of the protein is bound to the GPI anchoron the cell membrane, and thus the protein is immobilized on the cellmembrane. Then, the base portion of the GPI anchor is cleaved byphosphatidylinositol-dependent phospholipase C (PI-PLC). Then, theprotein cleaved from the cell membrane is incorporated into the cellwall so as to be immobilized on the cell surface layer and thus islocalized on the cell surface layer. Herein, “GPI anchor” refers to aglycolipid having ethanolamine phosphate−6 mannose α 1-2 mannose α 1-6mannose α 14 glucosamine α 1-6 inositol phospholipid, which is alsoknown as glycosylphosphatidylinositol (GPI), as the basic structure.

The GPI anchoring domain is generally positioned at or near theC-terminus of the cell surface layer-localized protein. For example, inaddition to the GPI anchor attachment recognition signal sequence, thereare four sugar chain-binding sites in the sequence encoding 320 aminoacid residues from the C-terminus of α-agglutinin. These sugarchain-binding sites and polysaccharides constituting the cell wall arecovalently bonded after the GPI anchor is cleaved by PI-PLC, so that theC-terminal sequence portion of α-agglutinin is bonded to the cell walland thus the α-agglutinin is retained on the cell surface layer.

The inventors succeeded in expressing lipase on the cell surface layer,utilizing such a GPI anchoring domain (Japanese Laid-Open PatentPublication No. 11-290078). More specifically, the structural gene oflipase was placed upstream of the DNA encoding the GPI anchoring domain,and a secretion signal sequence was placed further upstream, so that thelipase was expressed on the cell surface layer such that the N-terminusthereof was outside the cell.

DISCLOSURE OF INVENTION

The thus expressed protein can exhibit sufficient activity in the cellsurface layer, as long as it has the active center on the N-terminalportion. However, when the active center is on the C-terminal portion,the active center is too dose to the cell surface layer so that sterichindrance may occur and prevent sufficient activities from beingexhibited.

The inventors have examined various methods for expressing a protein onthe cell surface layer. As a result, it was unexpectedly found that evenif the function of the GPI anchoring domain, which had been consideredto be essential, was lost, the GPI anchor protein could be retained onthe cell surface layer, and thus the present invention was achieved. Inother words, the present invention has made it possible that if at leastthe flocculation functional domain of the GPI anchor protein iscontained, a desired protein can be expressed on a cell surface layer byconstructing a plasmid that allows the desired protein to be expressedon either the N-terminus or the C-terminus or both the N-terminus andthe C-terminus.

The present invention provides a cell surface layer-binding proteincomprising a sugar chain-binding protein domain, in which an additionalprotein is fused to at least an N-terminus or a C-terminus of the sugarchain-binding protein domain.

In a preferable embodiment, the sugar chain-binding protein domain is aportion including at least a flocculation functional domain of a GPIanchor protein.

In a preferable embodiment, the GPI anchor protein is a flocculationprotein.

In a preferable embodiment, the flocculation protein is a proteinselected from the group consisting of FLO1, FLO2, FLO4, FLO5, FLO9,FLO10 and FLO11.

In another preferable embodiment, the additional protein is fused to theN-terminus of the sugar chain-binding protein domain.

In a more preferable embodiment, the additional protein is fused to theC-terminus of the sugar chain-binding protein domain.

In a preferable embodiment, the additional protein is lipase.

In a preferable embodiment, the additional protein is an antibody.

In a preferable embodiment, the same or different additional proteinsare fused to the N-terminus and the C-terminus of the sugarchain-binding protein domain.

The present invention also provides DNA encoding the above-describedcell surface layer-binding protein.

The present invention further provides a plasmid comprising theabove-described DNA.

The present invention further provides a cell wherein the DNA or theplasmid is introduced, and the additional protein is expressed on a cellsurface layer.

In a preferable embodiment, the cell is yeast.

In a preferable embodiment, the additional protein is an enzyme.

The present invention also provides a method for using theabove-described cell wherein the additional protein is contacted with asubstrate, so that the substrate is bound or the substrate is convertedinto another substance.

The present invention also provides an enzyme-containing agentcomprising the above-described cell.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view of construction of a plasmid pWIFSpmROL.

FIG. 2 is a graph showing the growth of various yeasts having lipase ona cell surface layer and the lipase activities in the yeasts.

FIG. 3 is a graph showing the methanolysis activities of yeastMT8-1-short expressing a short-type FLO1-lipase and yeast MT8-1-longexpressing a long-type FLO1-lipase.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, “cell surface layer-binding protein” refers toa fused protein that includes at least a sugar chain-binding proteindomain and another protein and is immobilized on a cell surface layer bythe sugar chain-binding protein domain.

In the present invention, “sugar chain-binding protein domain” refers toa domain that has a plurality of sugar chains and can stay on a cellsurface later due to interaction and/or entwinement between these sugarchains and the sugar chains in the cell wall. Examples thereof includesugar chain-binding sites of lectin, lectin-like protein and the likes.A typical example thereof is a flocculation functional domain of the GPIanchor protein. Here, “flocculation functional domain of the GPI anchorprotein” refers to a domain that is positioned on the N-terminal portionof the GPI anchoring domain, has a plurality of sugar chains, and isconsidered to participate in flocculation.

In the present invention, “GPI anchor protein” generally refers to acell surface layer localized protein that can be bound to a cellmembrane via the GPI anchor present on the cell membrane. The GPI anchorprotein has a secretion signal sequence at the N-terminus and the GPIanchoring domain at the C-terminus. Examples of the GPI anchor proteininclude, but are not limited to, a flocculation protein of yeast(α-agglutinin, a-agglutinin, and FLO proteins), and alkalinephosphatase. In particular, FLO proteins, such as FLO1, FLO2, FLO4,FLO5, FLO9, FLO10 and FLO11 can be preferably used as the flocculationprotein in the present invention.

In the present invention, “GPI anchoring domain” refers to a domainconstituting from a cell wall anchoring domain and a GPI anchorattachment recognition signal sequence. In general, the GPI anchoringdomain is positioned at or near the C-terminus of a cell surfacelayer-localized protein. For example, in an α-agglutinin of yeast, theGPI anchoring domain is a sequence of 320 amino acids from theC-terminus.

The GPI anchor attachment recognition signal sequence of the GPIanchoring domain is a portion that recognizes the GPI anchor on a cellmembrane. After the GPI anchor attachment recognition signal sequence atthe C-terminus is cleaved, the GPI anchor is bound to a newly projectedC-terminus. Then, the base portion of the bound GPI anchor is cleaved byPI-PLC, and thus the protein is detached from the cell membrane andincorporated into the cell wall. Here, the sugar chains present in thecell wall anchoring domain are bound to the sugar chains in the cellwall and thus the protein is immobilized on the cell surface layer.

The cell surface layer-binding protein of the present invention includes(1) a fused protein in which an additional protein is fused to theN-terminus of the sugar chain-binding protein domain, (2) a fusedprotein in which an additional protein is fused to the C-terminus of thesugar chain-binding protein domain, and (3) a fused protein in which thesame or different proteins are fused both to the N-terminus and theC-terminus of the sugar chain-binding protein domain. The additionalprotein may be fused directly to the sugar chain-binding protein domainor may be fused thereto via a linker. The secretion signal sequence isfused to the N-terminus of the N-terminus-bound additional protein.

There is no limitation regarding the additional protein that constitutesthe cell surface layer-binding protein of the present invention, but aprotein that is not originally localized on the cell surface layer andis placed for the purpose of immobilization on the cell surface layer ispreferable. Examples thereof include secretory proteins and antibodies.Examples of secretory proteins include lipase, amylases (glucoamylase,α-amylase and the like), cellulases, fluorescent protein, protein A andderivatives thereof.

It can be determined whether these enzymes are fused on the N-terminusor the C-terminus, depending on their characteristics. In particular, aprotein having the active center on the C-terminal region such as lipaseis preferably fused to the C-terminus of the sugar chain-binding proteindomain. This protein may be directly fused to the C-terminus of thesugar chain-binding protein domain, or may be fused, for example, via apart of the GPI anchoring domain.

It is preferable that the protein is fused between the secretion signalsequence and the sugar chain-binding protein domain when it is fused tothe N-terminus. Furthermore, for example, a part of the GPI anchoringdomain may be contained on the C-terminus of the sugar chain-bindingprotein domain. However, in this case, the GPI anchor attachmentrecognition signal is not contained, and therefore it is not via the GPIanchor that an expressed fused protein is immobilized on the cellsurface layer.

The secretion signal sequence is an amino acid sequence that is bound tothe N-terminus of a protein (secretory protein) that is secreted outsidea cell (including periplasm) and contains a large number of hydrophobicamino acids. In general, the secretion signal sequence is removed whenthe secretory protein passes across a cell membrane from the inside of acell and is secreted outside the cell.

In the present invention, any secretion signal sequence can be used, aslong as it can lead an expressed fused protein to the cell membrane, andthere is no limitation regarding its origin. For example, as thesecretion signal sequence, the secretion signal sequence ofglucoamylase, the signal sequence of α- or a-agglutinin of yeast, thesecretion signal sequence of lipase can be preferably used. A part of orthe entire secretion signal sequence and/or the prosequence thereof canremain in the N-terminus, as long as it does not affect the activity ofthe additional protein fused with the cell surface layer-bindingprotein.

In this specification, “lipase” refers to a protein (enzyme) having anactivity that can allow a fatty acid to be released from oils and fats.Herein, “oils and fats” refers to glycerol bound to a fatty acid. Thereis no particular limitation regarding the origin of the lipase, as longas it has such an activity. In general, lipase derived frommicroorganisms, plants, and animals (e.g., porcine pancreas) can beused. Furthermore, lipase can be 1,3-specific or can be nonspecific. Forexample, biodiesel fuel can be produced from waste oil by expressinglipase on the cell surface layer of flocculent yeast.

In this specification, “amylases” refer to enzymes that hydrolyzestarch. Typical examples thereof include glucoamylase and α-amylase, andfurther include β-amylase and isoamylase.

In this specification, “glucoamylase” refers to an exo-type hydrolasethat cleaves glucose units from the non-reducing end of starch. There isno particular limitation regarding its origin, as long as it has such anactivity. In general, glucoamylase derived from fungi such as Rhizopusand Aspergillus can be used. For example, as described in Ueda et al.(Appl. Environ. Microbiol. 63:1362–1366 (1997)), glucoamylase derivedfrom Rhizopus oryzae can be preferably used. For example, ethanol can beproduced efficiently in the presence of starch by expressingglucoamylase on the surface layer of yeast.

In the present invention, “α-amylase” refers to an endo-type enzyme thathydrolyses only α1,4-glucoside bond of starch. There is no particularImitation regarding its origin, as long as it has such an activity. Forexample, α-amylase derived from animals (saliva, pancreas, etc.), plants(malt, etc) and microorganisms are used. For example, ethanol can beproduced efficiently in the presence of starch by expressing α-amylaseon a yeast surface layer. Alcoholic fermentation can be performedefficiently when α-amylase is expressed in combination with theglucoamylase.

“Cellulases” generally refers to endo β1,4-glucanase, but in the presentinvention, cellulase refers to a group of enzymes that is producedtogether with β1,4-glucanase, cleave a β1,4-glucoside bond and produceglucose from cellulose (e.g., cellobiohydrolase, β-glucosidase).Examples of cellulases include endo β1,4-glucanase, cellobiohydrolase,β-glucosidase, and carboxymethyl cellulase. There is no particularlimitation regarding its origin, as long as it has such an activity. Forexample, cellulases derived from microorganisms can be preferably used.

Alcoholic fermentation can be performed efficiently from a woodymaterial such as paper, pulp or their waste materials by expressingcellulase alone or in combination on a yeast surface layer.

In the present invention, there is no particular limitation regardingcells in which a protein is expressed on its cell surface layer, as longas they have a cell wall such as bacteria, fungi, or plant cells.Preferably, yeast can be used.

The cell used in the present invention is a cell that has beentransformed by introducing DNA such that a desired protein is displayedon the cell surface layer. The DNA to be introduced includes sequencesencoding at least the secretion signal sequence, the sugar chain-bindingprotein domain and a desired protein. Furthermore, the DNA may include asequence encoding another desired protein.

The synthesis and ligating of the DNA containing the various sequencescan be performed by techniques that can be used generally by thoseskilled in the art.

It is desirable that the DNA is in the form of a plasmid. It ispreferable that the DNA is a shuttle vector for E. coli in that the DNAcan be amplified and obtained easily. The starting material of the DNAhas, for example, a replication origin (Ori) of a 2 μm plasmid of yeastand a replication origin of ColE1 and more preferably, it further has ayeast selective marker (e.g., a drug resistant gene, TRP, LEU2, etc.)and an E. coli selective maker (drug resistant gene, etc.). In order toexpress a structural gene of a protein, it is desirable that the DNAstarting material contains a so-called regulatory sequence forregulating the expression of this gene, such as operators, promoters,terminators and enhancers. Examples thereof include a GAPDH(glyceraldehyde 3′-phosphate dehydrogenase) promoter and a GAPDHterminator. Examples of a plasmid of such a starting material includeplasmid pYGA2270 or pYE22m containing a GAPDH (glyceraldehyde3′-phosphate dehydrogenase) promoter sequence and a GAPDH terminatorsequence, or a plasmid pWI3 containing UPR-ICL (an upstream region ofisocitrate lyase) sequence and Term-ICL (a terminator region ofisocitrate lyase) sequence.

Preferably, if DNA encoding a desired protein is inserted between theGAPDH promoter sequence and the GAPDH terminator sequence of the plasmidpYGA2270 or pYE22m, or between the UPR-ICL sequence and the Term-ICLsequence of the plasmid pWI3, a plasmid for yeast transformation can beconstructed. In the present invention, a multicopy plasmid pWIFS orpWIFL is used preferably, and for example, pWIFSpmROL or pWIFLpmROL isproduced.

As the yeast serving as a host, any yeast can be used, but flocculentyeast is preferable in that it is easily separated after a reaction, orin that it is easily immobilized so that sequential reactions can beperformed. Alternatively, when a flocculation functional domain is usedas the sugar chain-binding protein domain, strong flocculationproperties can be provided to any yeast.

Examples of flocculent yeast include Saccharomyces diastaticusATCC60715, Saccharomyces diastaticus ATCC60712, Saccharomyces cerevisiaeIF01953, Saccharomyces cerevisiae CG1945, and Saccharomyces cerevisiaeHF7C. Alternatively, new flocculent yeast can be constructed. Forexample, as shown in Example 1 described later, according to the methodof M. D. Rose et al. (Methods in Yeast Genetics, 1990, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), flocculent yeastYF207 and yeast having properties equal to those of this yeast can beobtained from diploid by conjugation of flocculent yeast ATCC60712 andnon-flocculent yeast W303-1B. The flocculent yeast YF207 strain obtainedby the inventors of the present invention has excellent plasmidstability, and a very high fermentative ability. Therefore, when arecombinant flocculent yeast YF207 strain is used in which eitherα-amylase or glucoamylase alone or both α-amylase and glucoamylase areexpressed on the cell surface layer, the efficiency of alcoholicfermentation from starch is very high.

The cells used in the method of the present invention can be obtained byintroducing the above-described DNA into cells. “Introduction of DNA”means that DNA is introduced into a cell and expressed therein. Exampleof the method for introducing DNA include transformation, transduction,transfection, cotransfection, and electroporation, and specific examplesthereof include a method employing lithium acetate and a protoplastmethod.

The DNA to be introduced may be in the form of a plasmid as describedabove, or may be incorporated into a chromosome by inserting DNA into agene of a host or causing homologous recombination between DNA and agene of a host.

The cell into which the DNA is introduced can be selected by a selectivemarker (e.g., TRP) and selected by measuring the activity of theexpressed protein. The fact that the protein is immobilized on the cellsurface layer can be confirmed by an immunological method employinganti-protein antibody and FITC labeled anti-IgG antibody.

The cells used in the present invention can be immobilized on a carrier.For example, in the case of yeast, immobilization thereon is convenientwhen it is used in repeated batch fermentation or continuousfermentation.

In this specification, “carrier” means a substance on which a cell canbe immobilized, and preferably a substance that is insoluble in water ora specific solvent. As a material of the carrier that can be used in thepresent invention, foams or resins, for example, polyvinyl alcohol,polyurethane foam, polystyrene foam, polyacrylamide, a polyvinyl formalresin porous substance, silicon foam, or a cellulose porous substanceare preferable. In view of exfoliation of cells whose growth andactivities are reduced or dead cells, porous carriers are preferable.The size of openings of pores of the porous substance depends on thecell, but a suitable size of the openings is such that the cells canenter sufficiently within the pores and be grown therein. A size of 50μm to 1000 μm is preferable but the size is not limited thereto.

The carrier may be of any shape. In view of the strength of the carrier,cultivation efficiency and the like, a spherical or cubical shape ispreferable. Preferable size is 2 mm to 50 mm in a diameter for aspherical carrier preferable and 2 mm to 50 mm in length of the side fora cubical carrier.

In this specification, “immobilization of a cell” means a state in whicha cell is not free, for example, a state in which a cell is bound orattached on a carrier, or incorporated into the inside of the carrier.For immobilization of a cell, methods commonly used by those skilled inthe art, such as a carrier binding method, a cross-linking method, or anentrapment method, can be used. Among these, for immobilization offlocculent yeast, a carrier binding method is most suitable. The carrierbinding method includes a chemically adsorption method by which thecells are adsorbed to ion-exchange resin or a physically adsorptionmethod.

For example, although the flocculent yeast that can be used in thepresent invention is immobilized on a carrier, it can grow thereon andhas the properties that the yeast is exfoliated spontaneously with thereduction in its activity. Therefore, the yeast bound to the carrier ischaracterized by maintaining a substantially constant number of liveyeast and having high activities. In view of these characteristics,physical adsorption is most preferable for binding to the carrier. Nospecial means is required for physical adsorption. Flocculent oradhesive cells are simply mixed with the porous carrier and cultivatedso that the cells enter the pores of the porous carrier and are adheredto the carrier.

In this specification, “flocculation properties” means the property thatcells such as yeast that are floating or dispersed in a liquid aggregateand form a mass (flocculate), and “adhesiveness” means the propertiesthat cells are adhered or bound to each other so as to form anaggregate.

In this specification, a “state in which the activity is reduced” meansa state in which the activity of an entire cell is weakened although thecell itself is not dead, or for example, a state in which the activityinvolved in flocculation is reduced or the activity in the DNA levelencoding an enzyme involved in flocculation is weakened so that cellscannot aggregate.

In the present invention, flocculent or adhesive yeast may be yeast thatis provided with the flocculation properties or adhesiveness byintroduction of a gene involved in flocculation or adhesion.

Examples of genes involved in flocculation or adhesion includesubstances involved in flocculation or adhesion, such as structuralgenes encoding chitin, lectin, etc. in yeast. Examples of genes involvedin the flocculation include genes such as FLO1 [J. Watari et al., Agric.Biol. Chem., 55:1547(1991), G. G. Stewart et al., Can. J. Microbiol.,23:441(1977), I. Russell et al., J. Inst. Brew., 86:120(1980), C. W.Lewis et al. J. Inst. Brew., 82:158(1976)], FLO5 [I. Russell et al., J.Inst. Brew., 85:95(1979)], and FLO8 [I. Yamashita et al., Agric. Biol.Chem., 48:131(1984)].

These genes involved in flocculation or adhesion are incorporated in theplasmid of the above-described starting material and are introduced in acell together with DNA that is designed such that a desired protein isdisplayed on the cell surface layer.

The thus obtained immobilized cell can be cultivated in a floated statewhile being attached onto a carrier or can be filled in a column or thelike and used as a so-called bioreactor. Even if the cells arerepeatedly cultivated and reacted continuously or by batch, cells whoseactivities are reduced or that are dead are exfoliated so that theactivity of the cells is not reduced, and the cells can be utilizedefficiently.

The cells of the present invention obtained in the above-describedmanner can be used for the purpose of binding a substrate or convertinga substrate to another substance by contacting the protein expressed onthe cell surface layer with the substrate. For example, when the proteinis an enzyme, the cell can be provided as an enzyme-containing agentthat includes a cell expressing that enzyme. More specifically, theenzyme-containing agent of the present invention includes a suspensioncontaining the cells of the present invention and a medium that canmaintain the cells, and those obtained by cryopreservation,lyophilization or low-temperature drying of the cells of the presentinvention.

Furthermore, the cells of the present invention can also be used as acombinatorial library by allowing each of various proteins to which amutation is introduced at random to be displayed on the surface layer ofanother cell. It is possible to select a clone having most suitableproperties for the purpose rapidly and easily. The “combinatoriallibrary” in this specification refers to a group of various mutants inwhich a mutation is introduced randomly at the gene level so that thevariety of the protein is expanded artificially.

Hereinafter, the present invention will be described by way of example,but is not limited thereto.

Example 1 Preparation of DNA Having the 5′ Region of FLO1 and theStructural Gene of Prolipase in this Order

A: Preparation of a Gene of the 5′ Region (Signal Sequence andFlocculation Functional Domain) of FLO1

A gene of FLO1 was obtained in the following manner. First, achromosomal DNA was extracted from S. cerevisiae ATCC60715. Then, usingthis as a template, PCR amplification was performed using nucleotidesequences described in SEQ ID NOS: 1 and 2 as primers, and the amplifiedproduct was digested with BamHI and BglII to give a BamHI-BglII fragment(3300 bp BamHI-BglII FLO1 fragment) having a length of about 3300 bp.This 3300 bp fragment seems to have a sequence of the 5′ region of theFLO1 (secretion signal sequence and a FLO1 flocculation functionaldomain).

B: Preparation of a Lipase Gene

A gene of Rhizopus oryzae lipase was prepared in the following manner.Briefly, first, a chromosomal DNA was extracted from R. oryzae IF04697.Then, using this as a template, PCR amplification was performed usingthe nucleotide sequences described in SEQ ID NOS: 3 and 4 as primers,and the amplified product was digested with BamHI and SalI to give aBamHI-SalI fragment (BamHI-SalI lipase fragment) having a length ofabout 1100 bp. This lipase fragment had the pro-sequence and the matureprotein sequence of lipase and is substantially the same sequence asthose described in the report of Beer et al. (Biochim Biophys Acta,1399: 173–180, 1998).

C: Construction of a Plasmid Having the 5′ Region of FLO1 and theStructural Gene of Prolipase in this Order

A plasmid having a desired DNA can be obtained by connecting the 5′region gene of FLO1 obtained in the above section A and the prolipasegene obtained in the above section B. In order to prepare a fusedprotein of a FLO1 derivative and lipase, the following procedure wasperformed. A schematic view of the preparation is shown in FIG. 1.

First, a multicopy plasmid pWI3 was digested with BamHI anddephosphorized, and then the 3300 bp BamHI-BglII FLO1 fragment obtainedin the above section A was inserted therein to give plasmid pWIFS. Then,this plasmid pWIFS was digested with BglII and XhoI, and the BamHI-SalIlipase fragment obtained in above section B was inserted therein to givepWIFSpmROL. The protein expressed from the gene that is inserted in thisplasmid pWIFSpmROL was designated “short-type FLO1-lipase”.

Example 2 Preparation of DNA Having the 5′ Region of FLO1 Including aCell Wall Anchoring Domain and the Structual Gene of Prolipase in thisOrder

A chromosomal DNA of FLO1 was extracted in the same manner as inExample 1. Using this as a template, PCR amplification was performedusing the nucleotide sequences described in SEQ ID NOS: 1 and 5 asprimers, and the amplified product was digested with BamHI and BglII togive a BamHI-BglII fragment (4500 bp BamHI-BglII FLO1 fragment) having alength of about 4500 bp. This 4500 bp fragment seems to have a sequenceof the 5′ region of the FLO1 (secretion signal sequence and a FLO1flocculation functional domain).

The same operation as in Example 1 was performed using the obtained 4500bp BamHI-BglII FLO1 fragment. More specifically, the 4500 bp fragment,instead of the 3300 bp BamHI-BglII FLO1 fragment of Example 1 wasinserted in pWI3 to give a plasmid pWIFL. Then, the BamHI-SalI lipasefragment was inserted in this plasmid pWIFL in the same manner as inExample 1 to give pWIFLpmROL. The protein expressed from the gene thatwas inserted in this plasmid pWIFLpmROL was designated “long-typeFLO1-lipase”

Example 3 Preparation of Yeast Having Lipase on a Cell Surface Layer

Using Saccharomyces diastaticus ATCC60712 (MATα leu2-4 3,112 his2 lys2sta1 FL08), which is a flocculent yeast, and W303-1B (MATα ura3-52 trp1Δ2 leu2-3,112 his3-11 ade2-1 can1-100), which is a non-flocculent yeast,according to the method of M. D. Rose et al. (described above),Saccharomyces cerevisiae YF207 (MATα ura3-52 trp1 Δ2 his ade2-1 can1-100sta1 FLO8), which is a new flocculent strain that is tryptophanauxotrophic, was obtained.

The plasmid pWIFSpmROL obtained in Example 1 and the plasmid pWIFLpmROLobtained in Example 2 were introduced in non-flocculent yeast S.cerevisiae MT8-1 (MATA ade his3 leu2 trp1 ura3) (Tajima et al., Yeast,1:67-77, 1985) or flocculent yeast YF207 by a lithium acetate methodemploying Yeast Maker (Clontech Laboratories, Inc., Palo Alto, Calif.).They were cultivated using a SD-W agar selective medium (6.7% Yeastnitrogen base w/o amino acids (manufactured by Difco Laboratories), 2%glucose and 2% agar powder) supplemented with L-tryptophan-free suitableamino acids and bases. The grown yeast was selected, and yeastexpressing the short-type FLO1-lipase was designated “MT8-1-short” and“YF207-short”, respectively, and yeast expressing the long-typeFLO1-lipase was designated “MT8-1-long” and “YF207-long”, respectively

The obtained yeast was cultivated in a SDC liquid medium, and separatedinto a medium and cells by centrifugation and each lipase activity wasmeasured. As the controls, yeast in which the plasmid pWI3 wasintroduced into S. cerevisiae MT8-1 was used. As a result, in thecontrols, the lipase activity was not observed in either the medium orthe cells. Although the lipase activity was substantially not observedin the culture supernatants of the transformants MT8-1-short,YF207-short, MT8-1-long, and YF207-long, the yeast cells themselves ofall the types had the lipase activities (FIG. 2). The lipase activitieswere measured using a Lipase Kit S (manufactured by DAINIPPONPHARMACEUTICAL CO., LTD.).

Example 4 Confirmation of the Function of Yeast Having Lipase on theCell Surface Layer

Regarding the yeast MT8-1-short and YF207-short which express theshort-type FLO1-lipase, and the yeast MT8-1-long and YF207-long whichexpress the long-type FLO1-lipase, obtained in Example 3, the growth ofthe cell of each type was measured based on the absorbance at awavelength of 600 nm. It was found that all of the types of yeast weregrown well in a medium to the same extent as that of the controls (datanot shown).

Then, the methanolysis activities of the yeast MT8-1-short expressingthe short-type FLO1-lipase, and the yeast MT8-1-long expressing thelong-type FLO1-lipase were measured. To each yeast collected from a 100ml medium in a 30 ml vial, a reaction mixture (2 ml of 0.1M phosphatebuffer solution (pH 7.0), 9.65 g of soybean oil, and 0.35g of methylalcohol) was added, and shaken at 35° C. 150 times per minute, and thenthe amount of produced methyl ester was measured by the method of Kaiedaet al. (J. Biosci. Bioeng., 88: 627–631, 1999). The results are shown inFIG. 3.

All the types of yeast produced more methyl ester than a conventionalyeast (yeast in which lipase is bound to the N-terminus of α-agglutininand expressed on the cell surface layer (Japanese Laid-Open PatentPublication No. 11-290078)). In particular, as shown in FIG. 3, in theyeast MT8-1-short (◯) expressing the short-type FLO1-lipase and theyeast MT8-1-long (Δ) expressing the long-type FLO1-lipase, theactivities in producing methyl ester were very high.

Furthermore, the flocculation abilities of these strains were measuredaccording to the method of Smit et al. (Smit et al., Appl. Environ.Microbiol., 58:3709–3714 (1992)). As a result, regarding the flocculentstrains, when compared with before the plasmid was introduced, thestrains expressing the long-type FLO1-lipase exhibited the flocculationability as strong as before the transformation. However, in the strainsexpressing the short-type FLO1-lipase, the flocculation ability wasweakened. This seems to be because the short-type FLO1-lipase has nocell wall anchoring domain, and instead the flocculation domain isembedded in the cell wall, so that the flocculation ability cannotsufficiently be exhibited.

Example 5 Construction of a Combinatorial Library of Randomly MutatedLipase and Utilization Thereof

A mutation introduction was performed with respect to the 1098 bp geneencoding the prosequence and the mature protein sequence (prolipase(ProROL) of 366 amino acids) of Rhizopus oryzae lipase obtained in thesection B of Example 1 at random by Error-prone PCR method such that amutation of about 3 bases (introduction of a mutation of one amino acidper molecule of ProROL) was contained, and thus variousmutation-introduced lipase genes were obtained. In the same manner asthe section C of Example 1, mutation-introduced lipase genes obtainedwere inserted in the plasmid pWIFS having the 5′ region of FLO1 to givea pWIFSProROL library. Each of these plasmids was introduced into theyeast YF207 strain in the same manner as in Example 3, and acombinatorial library of yeast consisting of 713 strains that displayedlipase having various mutations on the cell surface layer was produced.

A clone having transesterification activity was selected from theobtained yeast combinatorial library in the following manner. Thelibrary of 713 strains was cultivated on an SD plate (0.67% Yeastnitrogen base w/o amino acids, 0.5% glucose and 2% agar powder) forthree days. Then, a soft agar SD medium (SD medium+2.5% soybean oils,0.001% rhodamine B, 50% methanol and 0.8% agar powder) containing afluorochrome that was kept at 45° C. after autoclaving was layered overthis plate. One day later, the plate was irradiated with ultravioletrays and the clone emitting orange fluorescence around the cell wasselected to obtain clones (80 clones) that survived in the presence ofmethanol, which is the substrate of methanolysis.

Regarding the lipase activities of these clones, the initial rate of thetransesterification by methanolysis and the hydrolysis activities by aLipase Kit S (manufactured by DAINIPPON PHARMACEUTICAL CO., LTD.) weremeasured in the same manner as described in Example 4. Table 1 shows theresults of those strains among these strains, in which thetransesterification activities and the hydrolysis activities werechanged, compared with the strain (wild-type strains) expressing lipaseto which a mutation has not been introduced.

TABLE 1 Lipase activities transesterification hydrolysis reactionreaction transesterification/ mutant (g/l/minute) (IU/ml) hydrolysiswild-type 2.38 0.23 10.35 S334T 2.51 0.22 11.41 P2S/F293Y 2.04 0.34 6.00K36E 1.85 0.17 10.88 K326R 2.24 0.12 18.67 V56A/W224R 2.01 0.20 10.05Q225H/Q294L 0.94 0.14 6.71 S10P 1.53 0.17 9.00 N62D 1.63 0.20 8.15

The strain S334T had slightly higher transesterification activities thanthose of the wild-type strain. Furthermore, the relative values oftransesterification activities with respect to the hydrolysis activitiesindicate that the strain P2S/F293Y exhibited a high catalytic abilityspecific to hydrolysis, and the strain K326R exhibited a high catalyticability specific to transesterification. These results indicate that amutant suitable for the purpose can be screened by preparing acombinatorial library on the yeast cell surface layer.

INDUSTRIAL APPLICABILITY

The cell surface layer-binding protein of the present invention canexpress a desired protein on the cell surface layer. This desiredprotein can be expressed so as to have sufficient activities by,depending on the position of the active center, being fused to the sugarchain-binding protein domain on its opposite side. Therefore, inparticular, when expressing a protein having activities on theC-terminal portion on the cell surface layer, the present invention isvery useful. Furthermore, it is possible to express more proteinsefficiently by fusing the same or different proteins to both terminalsof the sugar chain-binding protein domain. A combinatorial library ofmodified proteins is prepared on the cell surface layer, utilizing suchefficient expression of proteins on the cell surface layer, so thatscreening can be performed easily.

1. A yeast cell surface layer-binding protein comprising a sugarchain-binding protein domain, in which an additional protein is fused toat least an N-terminus or a C-terminus of the sugar chain-bindingprotein domain, wherein the sugar chain-binding protein domain includesa flocculation functional domain of a GPI anchor protein but does notinclude a GPI anchor attachment recognition signal sequence present in aC-terminal portion of the GPI anchor protein, wherein the GPI anchorprotein is a flocculation protein of yeast.
 2. The yeast cell surfacelayer-binding protein according to claim 1, wherein the flocculationprotein is a protein selected from the group consisting of FLO1, FLO2,FLO4, FLO5, FLO9, FLO10 and FLO11.
 3. The yeast cell surfacelayer-binding protein according to claim 1, wherein the additionalprotein is fused to the N-terminus of the sugar chain-binding proteindomain.
 4. The yeast cell surface layer-binding protein according toclaim 1, wherein the additional protein is fused to the C-terminus ofthe sugar chain-binding protein domain.
 5. The yeast cell surfacelayer-binding protein according to claim 1, wherein the same ordifferent additional proteins are fused to the N-terminus and theC-terminus of the sugar chain-binding protein domain.
 6. DNA encodingthe yeast cell surface layer-binding protein according to claim
 1. 7. Aplasmid comprising the DNA according to claim
 6. 8. A yeast cell whereinthe DNA according to claim 6 or the plasmid according to claim 7, isintroduced and the additional protein is expressed on a cell surfacelayer.
 9. The yeast cell according to claim 8, wherein the additionalprotein is an enzyme.
 10. A method for using the yeast cell according toclaim 8, comprising contacting the additional protein with a substrate,so that the substrate is bound thereto or the substrate is convertedinto another substance.
 11. An enzyme-containing agent comprising theyeast cell according to claim
 9. 12. DNA encoding the yeast cell surfacelayer-binding protein according to claim
 2. 13. DNA encoding the yeastcell surface layer-binding protein according to claim
 3. 14. DNAencoding the yeast cell surface layer-binding protein according to claim4.
 15. DNA encoding the yeast cell surface layer-binding proteinaccording to claim 5.